Mycobacterium tuberculosis, the causative agent of tuberculosis, has been causing disease in humans since the beginning of civilization (8). Despite more than 50 years of vaccine and antibiotic development, it has been estimated that 225 million new cases of tuberculosis will arise between the years 1998 and 2030, with 79 million tuberculosis-related deaths (25). One of the challenges in treating this disease is the wide-spread development of multidrug-resistant tuberculosis (MDR-TB), defined as an infection that does not respond to treatment with either first-line drug isoniazid or rifampicin (9). The development of MDR-TB has resulted in an increased emphasis on the use of second-line drugs to treat these infections. One of these second-line drugs is capreomycin (CMN), a collection of four structurally related peptide antibiotics (FIG. 1). The importance of CMN for the treatment of MDR-TB is reflected in this drug being included on the World Health Organization's “List of Essential Medicines” (36). There is additional interest in CMN because of the recent finding that this drug is bactericidal against nonreplicating M. tuberculosis, suggesting the potential use of CMN to treat latent tuberculosis infections (15). While of most interest for treatment of tuberculosis, capreomycin is known in the art to be more generally active against a number of gram-positive and gram-negative bacteria.
CMN and a structural analog viomycin (VIO) (FIG. 1) (see also U.S. Pat. No. 7,326,782 for details of the VIO cluster and biosynthesis thereof) disrupt the growth of Mycobacterium spp. by interfering with the function of the ribosome (22-24, 37-43). This conclusion is based on the isolation and characterization of resistant strains along with in vitro analysis of ribosome binding and disruption of peptide synthesis. There were two surprising results from these studies that emphasize the distinct mechanism of action of CMN and VIO. First, resistance to VIO in M. smegmatis was conferred by mutations in either the 16S or 23S rRNA, suggesting this antibiotic interferes with the function of both ribosomal subunits (41). The second result was the recent finding that M. tuberculosis resistance to CMN and VIO can also arise from a mutation in the gene tlyA (22). Subsequent analysis determined that the TlyA enzyme likely catalyzes methylation of both the 16S and 23S rRNA, and these methylations are essential for CMN and VIO sensitivity, possibly forming part of the binding site of these antibiotics (18). Thus, resistance to CMN and VIO can arise from point mutations in the 16S or 23S rRNA or from the loss of modifications to these rRNAs.
While the CMN and VIO resistance mechanisms in Mycobacterium spp. involve mutations in the rRNA or a gene coding for an rRNA modifying enzyme, the bacteria that naturally produce these antibiotics are proposed to have resistance mechanisms that are independent of ribosome modification (2, 28, 33). VIO resistance by Streptomyces sp. strain ATCC11861 (previously known as Streptomyces vinaceus) occurs via antibiotic inactivation by Vph, a VIO phosphorylase (2). CMN, while commonly referred to as a single molecule, is actually a mixture of four structural derivatives (FIG. 1). The structural differences between these derivatives are particularly relevant in the context of resistance genes carried by the producing bacterium. For example, a gene coding a homolog of Vph was isolated from the CMN-producer Saccharothrix mutabilis subsp. capreolus (previously Streptomyces capreolus), but this only conferred resistance to CMN IA and IIA, leaving CMN IB and IIB active. This selectivity is due to Cph catalyzing the phosphorylation of the hydroxyl group of residue 2 of CMN IA and IIA that is absent from CMN IB and IIB (FIG. 1) (28, 33). A second gene, cac, was identified and conferred resistance to all four derivatives of CMN (28, 33). The coded enzyme is proposed to catalyze the acetylation of the α-amino group of residue 1 of the cyclic pentapeptide core, thereby inactivating CMN. Consistent with this proposal, Cac did not confer resistance to VIO because of the β-lysine attached to the α-amino group of residue one of VIO.
CMN and VIO are peptide antibiotics with subtly different cyclic pentapeptide cores that can be decorated by carbamoylation, hydroxylation, or acylation with β-lysine (FIG. 1). A series of precursor labeling studies have been performed on CMN and VIO to investigate how these unusual antibiotics are assembled by the producing bacteria (4, 5, 12, 35). We outlined a biosynthetic mechanism for the assembly of VIO using these labeling experiment results in combination with bioinformatic and biochemical analysis of the VIO biosynthetic gene cluster and the enzymes it codes (19, 34) (U.S. Pat. No. 7,326,782). We predict that CMN biosynthesis follows similar mechanisms, but with some differences to account for the structural differences between these antibiotics.
Our interests in deciphering how this family of antibiotics is biosynthesized are in two areas. First, we address the basic biological question of how these unusual cyclic peptides, consisting of rare nonproteinogenic amino acids, are assembled. Second, we explore harnessing of the biosynthetic machinery of CMN and VIO production to generate new structural derivatives of these antibiotics through the use of metabolic engineering. Here we present the isolation and sequencing of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus strain ATCC 23892. Bioinformatics analysis of this gene cluster provides a molecular blueprint for CMN biosynthesis and explains the structural differences between CMN and VIO. The integration of this biosynthetic gene cluster into the chromosome of Streptomyces lividans 1326 resulted in the heterologous production of CMN by this naturally non-producing bacterium. This is a significant finding because S. mutabilis subsp. capreolus has proven intractable to genetic manipulation (27). Thus, metabolic engineering of CMN biosynthesis was not previously possible in the natural producer. The results presented here circumvent this problem. Finally, while previous work suggested that S. mutabilis subsp. capreolus does not alter its ribosomes to become resistant to CMN, we present data that strongly suggests ribosome modification is a natural CMN resistance mechanism for this bacterium.